While iron is an essential cofactor for many proteins, its favorable chemical properties can also promote toxic side reactions that damage macromolecules. Failure to maintain proper iron homeostasis can lead to anemia or iron overload disorders, as well as increased susceptibility to infection. Cellular iron homeostasis is maintained by the coordinate posttranscriptional regulation of gene products responsible for iron uptake, release, utilization, and storage. When cellular free iron availability is low, Iron Regulatory Proteins 1 and 2 (IRP1 and 2) bind Iron Response Elements (IREs) within the 5' or 3' untranslated regions of these mRNAs to affect their subsequent translation or stability. When cellular free iron availability is high, IRP1 assembles an iron-sulfur cluster, causing the protein to lose its affinity for IREs, while IRP2 is preferentially ubiquitinated and degraded by the proteasome. However, the underlying mechanism of how the cell senses iron levels and subsequently regulates IRP2 degradation is poorly understood and has proven to be extremely controversial. To address the outstanding questions related to cellular iron sensing and IRP regulation, a cell-based siRNA screen was performed to identify E3 ubiquitin ligases that regulate IRP2 stability. The top candidate from that screen has been selected for further characterization. Preliminary studies indicate that the E3 ubiquitin ligase complex containing the FBXL5 protein, SCFFBXL5, directly targets IRP2 for proteasomal degradation when cellular free iron availability is high. The stability of FBXL5 itself is regulated, accumulating under iron and oxygen replete conditions and targeted for degradation upon iron depletion. FBXL5 appears to contain an iron- and oxygen-binding hemerythrin domain that acts as a ligand-binding regulatory switch mediating FBXL5's differential stability. These observations suggest a direct mechanistic link between iron sensing via a hemerythrin domain, FBXL5 accumulation, IRP2 regulation, and cellular responses to maintain mammalian cellular iron homeostasis. The broad objectives of this proposal are to validate the role of SCFFBXL5 in the regulation of IRP2, characterize the molecular mechanisms responsible for FBXL5's function(s) and regulation, and investigate the importance of FBXL5 to the maintenance of mammalian iron homeostasis in vivo. Specifically, this proposal aims to (1) map and characterize the functional and regulatory domains of FBXL5 using cultured cells and in vitro reconstitution assays, (2) investigate the ligand binding properties of the hemerythrin sensor using a variety of biophysical techniques, (3) identify additional factor(s) that regulate the iron-dependent stability of the hemerythrin domain, and (4) generate and characterize mice lacking FBXL5 expression. Together these studies will greatly inform our understanding of mammalian iron homeostasis and may provide new insights for treatment of related human diseases.